Bead Incubation and Washing on a Droplet Actuator

• US 20110203930A1
• Filed: 04/07/2011
• Published: 08/25/2011
• Est. Priority Date: 04/18/2006
• Status: Active Grant

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Sista, Ramakrishna, Pamula, Vamsee K., Sudarsan, Arjun
• Granted Patent: US 8,637,324 B2
• Time in Patent Office: Days
• US Class Current: 204/557
• CPC Class Codes: B01F 31/65 the materials to be mixed being directly submitted to a pulsating movement, e.g. by means of an oscillating piston or air column
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01F 33/3031 using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0424 Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 2400/0436 acoustic forces, e.g. surface acoustic waves [SAW]
• CPC Class Codes: B01L 2400/0442 : thermal energy, e.g. vaporisation, bubble jet
• CPC Class Codes: B01L 3/502715 : characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 : specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502784 : specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: B01L 3/502792 : for moving individual droplets on a plate, e.g. by locally altering surface tension
• CPC Class Codes: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B01L 7/525 : with physical movement of samples between temperature zones
• CPC Class Codes: B03C 1/02 : acting directly on the substance being separated
• CPC Class Codes: B03C 1/288 : disposed at the outer circumference of a recipient
• CPC Class Codes: B03C 1/30 : Combinations with other devices, not otherwise provided for
• CPC Class Codes: B03C 2201/18 : Magnetic separation whereby the particles are suspended in a liquid
• CPC Class Codes: B03C 2201/26 : for use in medical applications
• CPC Class Codes: B03C 5/005 : Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength B03C5/02 takes precedence; electrophoresis B01D57/02
• CPC Class Codes: C12N 15/1013 : by using magnetic beads
• CPC Class Codes: G01N 2035/1046 : Levitated, suspended drops
• CPC Class Codes: G01N 33/54333 : Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
• CPC Class Codes: G01N 33/561 : Immunoelectrophoresis
• CPC Class Codes: Y10T 436/11 : Automated chemical analysis
• CPC Class Codes: Y10T 436/25 : including sample preparation
• CPC Class Codes: Y10T 436/25625 : Dilution
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

The present invention relates to bead incubating and washing on a droplet actuator. Methods for incubating magnetically responsive beads that are labeled with primary antibody, a sample (i.e., analyte), and secondary reporter antibodies on a magnet, on and off a magnet, and completely off a magnet are provided. Also provided are methods for washing magnetically responsive beads using shape-assisted merging of droplets. Also provided are methods for shape-mediated splitting, transporting, and dispensing of a sample droplet that contains magnetically responsive beads. The apparatuses and methods of the invention provide for rapid time to result and optimum detection of an analyte in an immunoassay.

Claims

1. A method of incubating droplets having magnetically responsive beads therein, the method comprising:

(a) providing a droplet actuator comprising;

(i) droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface; and

(ii) a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet'"'"'s magnetic field capable of substantially attracting magnetically responsive beads in the droplet;

(b) positioning a droplet having magnetically responsive beads therein at a location on the droplet operations surface within the first region of the magnetic field to form a concentration of beads in the droplet;

(c) transporting the droplet through activation of selected droplet operations electrodes away from the first region of the magnetic field, thereby resuspending the magnetically responsive beads in the droplet;

(d) operating the droplet operations electrodes to cause the droplet to split into two droplets, thereby redistributing the magnetically responsive beads; and

(e) operating the droplet operations electrodes to merge the two droplets into a single droplet.

2. The method of claim 1, wherein at least one of steps (c), (d), and (e) is conducted with the magnetically responsive beads positioned at a locus of the droplet operations surface sufficiently near the magnet such that they are actively attracted to the first region of the magnetic field.

3. The method of claim 1, wherein at least one of steps (c), (d), and (e) is conducted with the magnetically responsive beads positioned at a locus of the droplet operations surface at a sufficient distance from the magnetic field such that they are not substantially aggregated by the first region of the magnetic field.

4. The method of claim 1, wherein the droplet transported has a footprint approximately one of one, two, or three times the area of a single droplet operations electrode.

5. The method of claim 1, wherein the droplet comprises a sample comprising a target substance, and the magnetically responsive beads comprise beads having an affinity for the target substance.

6. The method of claim 5, wherein the target substance is at least one of cells, protein, nucleic acid, and an antigen.

7. The method of claim 1, wherein the transporting, splitting, and merging operations are repeated a plurality of times.

8. The method of claim 1, wherein in step (b) the concentration of magnetically responsive beads occurs at an edge of the droplet adjacent the magnet.

9. The method of claim 1, wherein in step (c), because of the activation of selected droplet operation electrodes, the droplet becomes elongated as a result of the transporting.

10. The method of claim 1, wherein in step (e) the merging occurs within a region where the magnet is located.

11. A method of incubating droplets having magnetically responsive beads therein, the method comprising:

(a) providing a droplet actuator comprising;

(i) droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface; and

(ii) a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet'"'"'s magnetic field capable of substantially attracting magnetically responsive beads in the droplet;

(b) positioning a droplet having magnetically responsive beads therein at a location within the first region of the magnetic field of the magnet to form a concentration of beads in the droplet;

(c) transporting the droplet through activation of selected droplet operations electrodes away from the first region of the magnetic field of the magnet to resuspend the magnetically responsive beads in the droplet;

(d) operating the droplet operations electrodes to cause the droplet to elongate and then split into two droplets at a location away from the magnet; and

(e) operating the droplet operations electrodes to merge the two droplets into a single droplet at a location away from the magnet, whereby the transporting, splitting, and merging comprise an incubation cycle.

12. The method of claim 11, wherein in step (b) the concentration of magnetically responsive beads occurs at an edge of the droplet adjacent the magnet.

13. The method of claim 11, wherein the droplet transported has a footprint approximately three times the area of a single droplet operations electrode.

14. The method of claim 11, wherein the droplet comprises a sample comprising a target substance, and the magnetically responsive beads have an affinity for the target substance.

15. The method of claim 14, wherein the target substances are at least one of cells, protein, nucleic acid, and an antigen.

16. The method of claim 11, wherein the transporting, splitting, and merging operations are repeated a plurality of times.

17. A method of incubating droplets having magnetically responsive beads therein, the method comprising:

(a) providing a droplet actuator comprising;

(i) droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface; and

(ii) a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet'"'"'s magnetic field capable of substantially attracting magnetically responsive beads in the droplet;

(b) positioning a droplet having magnetically responsive beads therein on a droplet operations electrode, the droplet having a footprint approximately two times the area of a single droplet operations electrode;

(c) transporting the droplet through activation of selected droplet operations electrodes in one direction in a manner elongating the droplet; and

(d) operating the droplet operations electrodes in a manner to cause the droplet to be transported in an opposite direction to cause mixing and incubation within the droplet.

18. The method of claim 17, wherein the droplet comprises one part magnetically responsive beads and one part sample.

19. The method of claim 17, wherein the droplet comprises one part magnetically responsive beads and one part secondary reporter antibodies.

20. The method of claim 17, conducted substantially outside of the first region of the magnetic field.

21. The method of claim 18, wherein the sample comprises a target substance, and the magnetically responsive beads have an affinity for the target substance.

22. The method of claim 21, wherein the target substance is at least one of cells, protein, nucleic acid, and an antigen.

23. A method of washing magnetically responsive beads for separating and removing unbound material, the method comprising:

(a) providing a droplet actuator comprising;

(i) droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface; and

(ii) a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet'"'"'s magnetic field capable of substantially attracting magnetically responsive beads in the droplet;

(b) positioning a droplet having magnetically responsive beads therein to have a first region of the droplet within the first region of the magnetic field to form a concentration of beads;

(c) at another end of the magnetic field, positioning a wash buffer droplet such that a first region of the wash buffer droplet is within the first region of the magnetic field;

(d) operating the droplet operations electrodes to merge the droplet and the wash droplet to cause redistribution of beads;

(e) operating the droplet operation electrodes to cause the merged droplet to partially move away from the magnet, and to cause beads in the droplet to concentrate in the merged droplet; and

(f) operating the droplet operations electrodes to split the merged droplet to form a supernatant droplet containing unbound reagents.

24. The method of claim 23, wherein in step (b) the concentration of magnetically responsive beads in the droplet occurs at an edge of the droplet adjacent the first region of the magnetic field, and in step (e) the concentration of beads in the merged droplet occurs at an edge of the merged droplet located at the magnet.

25. The method of claim 23, wherein in step (b) the droplet is positioned to have a first region on a droplet operations electrode within the first region of the magnetic field and a second region of the droplet on an adjacent droplet operations electrode.

26. The method of claim 23, wherein in step (c) the first region of the wash buffer droplet is positioned on a droplet operations electrode within the first region of the magnetic field, and the second region of the wash droplet is on an adjacent droplet operations electrode.

27. The method of claim 23, wherein the wash droplet and the sample droplet each have a footprint approximately two times the area of one droplet operations electrode.

28. The method of claim 23, further comprising repeating the method a plurality of times to provide for sufficient removal of unbound material.

29. A method of resuspending magnetically responsive beads between wash cycles, the method comprising:

(a) providing a droplet actuator comprising;

(i) droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface; and

(ii) a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet'"'"'s magnetic field capable of substantially attracting magnetically responsive beads in the droplet;

(b) positioning a droplet having magnetically responsive beads therein at a location partially overlapping the first region of the magnetic field;

(c) transporting the droplet through activation of selected droplet operations electrodes away from the first region of the magnetic field;

(d) operating the droplet operations electrodes to cause the droplet to move towards the first region of the magnetic field; and

(e) repeating steps (c) and (d) to cause sufficient resuspension of beads such that unbound material may be effectively removed in subsequent wash cycles.

30. The method of claim 29, wherein step (b) is conducted by positioning the droplet at a location partially overlapping a droplet operations electrode located within the first region of the magnetic field.

31. The method of claim 29, further comprising conducting a wash cycle after resuspension of the beads to remove unbound material.

32. The method of claim 29, wherein the droplet comprises a sample comprising a target substance, and the magnetically responsive beads comprise beads having an affinity for the target substance.

33. The method of claim 32, wherein the target substance is at least one of cells, protein, nucleic acid, and an antigen.

34. A droplet actuator device having a structure for conducting a bead washing protocol, the structure comprising:

(a) an array of droplet operations electrodes configured to provide a plurality of individual wash lanes, and a single waste lane intersecting each one of the plurality of wash lanes; and

(b) waste wells associated at the end of each one of the plurality of wash lanes, and at the end of the single waste lane.

35. A method of separating magnetically responsive beads from a droplet, the method comprising:

(a) providing a droplet actuator comprising;

(i) droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface; and

(ii) a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet'"'"'s magnetic field capable of substantially attracting magnetically responsive beads in the droplet;

(b) positioning a droplet having magnetically responsive beads therein within the first region of the magnetic field of the magnet to cause the magnetically responsive beads to be attracted to the magnet, and activating the droplet operations surface to cause the droplet to be circular in shape;

(c) operating the droplet operations surface to cause the droplet to move away from the first region of the magnetic field to form a concentration of magnetically responsive beads in the droplet, and the droplet operations surface being operated to cause the droplet to be transported away from the magnet one droplet operations electrode at a time, to cause the geometry of the droplet to be distorted; and

(d) continuing to transport the droplet away from the magnet to cause the concentration of magnetically responsive beads to break away from the droplet to result in a relatively small and highly concentrated magnetically responsive bead droplet left behind and held immobilized by the magnet.

36. The method of claim 35, wherein in step (c) the concentration of magnetically responsive beads occurs at an edge of the droplet adjacent to the magnet.

37. The method of claim 35, wherein in step (b) the droplet is positioned at a droplet operations electrode within the first region of the magnetic field of the magnet.

38. The method of claim 35, wherein in step (c) the droplet is caused to move from the droplet operations electrode within the first region of the magnetic field partially to an adjacent droplet operations electrode away from the magnet.

39. The method of claim 35, wherein the droplet comprises a sample comprising a target substance, and the magnetically responsive beads comprise beads having an affinity for the target substance.

40. The method of claim 39, wherein the target substance is at least one of cells, protein, nucleic acid, and an antigen.

41. A method of transporting magnetically responsive beads within droplets, the method comprising:

(a) providing a droplet actuator comprising;

(i) droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface; and

(ii) a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet'"'"'s magnetic field capable of substantially attracting magnetically responsive beads in the droplet;

(b) positioning a droplet having magnetically responsive beads located therein at a location wherein the droplet partially overlaps the magnet;

(c) operating the droplet operations electrodes to subject an edge of the droplet nearest the magnet to both a magnetic force from the first region of the magnetic field and an electrowetting force from the droplet operations electrodes, and to subject an edge of the droplet furthest from the magnet only to an electrowetting force, to cause the droplet to be transported away from the magnet while retaining the magnetically responsive beads within the droplet; and

(d) continuing to transport the droplet away from the magnet to cause the magnetically responsive beads to be redistributed within the droplet.

42. The method of claim 41, wherein the droplet comprises a sample comprising a target substance, and the magnetically responsive beads comprise beads having an affinity for the target substance.

43. The method of claim 42, wherein the target substance is at least one of cells, protein, nucleic acid, and an antigen.

44. A method of separating beads from a droplet onto a magnet, the method comprising:

(a) providing a droplet actuator comprising;

(i) droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface; and

(ii) a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet'"'"'s magnetic field capable of substantially attracting magnetically responsive beads in the droplet;

(b) positioning a droplet having magnetically responsive beads therein within the first region of the magnetic field of the magnet to cause the beads to be attracted to the magnet, and activating the droplet operations surface in a manner to cause the droplet to take an elongate shape;

(c) operating the droplet operations surface to activate one electrode at a time, to cause the droplet to move away from the magnet, and thereby cause the geometry of the droplet to be distorted; and

(d) continuing to operate the droplet operations surface to transport the droplet further away from the magnet and inactivating an electrode intermediate to the droplet to cause the droplet to split into a supernatant droplet and a smaller droplet that has the magnetically responsive beads therein.

45. The method of claim 44, wherein step (b) is conducted by positioning the droplet at a droplet operations electrode within the first region of the magnetic field of the magnet, and wherein an adjacent droplet operations electrode is activated to cause the droplet to take an elongate shape, and wherein step (c) is conducted by operating one electrode at a time.

46. The method of claim 44, wherein the sample droplet is of a size wherein its footprint is three times the area of a single droplet operations electrode.

47. The method of claim 44, wherein the droplet comprises a sample comprising a target substance, and the magnetically responsive beads comprise beads having an affinity for the target substance.

48. The method of claim 47, wherein the target substance is at least one of cells, protein, nucleic acid, and an antigen.

49. A droplet actuator structure for extracting DNA from a sample, the structure comprising:

(a) at least six on-actuator reservoirs interconnected for storing and dispensing different reagents onto the droplet actuator; and

(b) the reservoirs interconnected through paths of droplet operations electrodes, including at least two paths having magnets associated therewith, and a bead collection reservoir connected to the six on-actuator reservoirs through the droplet operations electrodes paths.

50. A method of extracting DNA from whole blood by using the droplet actuator structure of claim 49, the method comprising:

(a) dispensing a droplet of magnetically responsive beads suspended in a lysis buffer from a first of the six on-actuator reservoirs, and transporting the droplet through the droplet operations electrodes to a specific location having one of the magnets associated with the location, to hold the magnetically responsive beads within the droplet thereon;

(b) dispensing droplets of whole blood from a second reservoir and lysis buffer from a third reservoir into a fourth mixing reservoir to be mixed therein to form a cell lysate;

(c) dispensing droplets of the cell lysate across the magnetically responsive beads in succession and removing supernatant from the droplets while holding the magnetically responsive beads;

(d) dispensing wash droplets from at least a fifth reservoir to wash the magnetically responsive beads to remove cell debris; and

(e) eluting and collecting DNA captured on the magnetically responsive beads at the bead collection reservoir.

51. The method of claim 50, wherein the magnetically responsive beads have an affinity for the DNA in the whole blood.

52. A method of detecting a component in a sample, the method comprising:

(a) providing a droplet actuator comprising;

(i) droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface;

(ii) a magnet positioned related to the droplet operations surface such that a droplet controlled by one of more droplet operations electrodes may be positioned within or away from a first region of the magnet'"'"'s magnetic field capable of substantially attracting magnetically responsive beads in the droplet; and

(iii) a wash reservoir at one end of the arrangement of droplet operations electrodes;

(b) positioning a droplet having magnetically responsive beads located therein, the magnetically responsive beads being coated with an antibody having an affinity for a specific target antigen, away from the magnet;

(c) operating the droplet operations surface in a manner to repeatedly transport the droplet back and forth, away from the magnet, in a manner to provide sufficient resuspension and mixing of the magnetically responsive beads for antibody and antigen binding;

(d) operating the droplet operations surface in a manner to transport the droplet to a location within the first region of the magnetic field, and splitting off a supernatant droplet from the droplet by selectively operating the droplet operations surface, and retaining the magnetically responsive beads at the magnet;

(e) operating the droplet operations electrodes to transport a reagent droplet to the droplet operations electrode in the first region of the magnetic field to merge the reagent droplet with the droplet containing the magnetically responsive beads, and transporting the merged droplet back and forth, at a location away from the magnet, to cause incubation; and

(f) transporting the merged droplet through operation of the droplet operations electrodes to the droplet operations electrode at the magnet and splitting off a supernatant droplet through operation of the droplet operations electrodes.

53. The method of claim 52, further comprising conducting the method to detect cytokine.

54. The method of claim 52, further comprising transporting a wash droplet from the wash reservoir through operation of the droplet operations electrodes, across the magnet to cause merging of the droplets, and splitting to yield a washed droplet with beads therein, and discarding a resultant supernatant droplet.

55. The method of claim 52, further comprising transporting at least one reagent droplet to the droplet operations electrode in the first region of the magnetic field, and merging the reagent droplet with the droplet through operation of the droplet operations electrodes, incubating the merged droplet away from the magnet, transporting the merged droplet following incubation to the droplet operations electrode in the first region of the magnetic field, and splitting off a supernatant droplet.

56. The method of claim 52, further comprising washing the droplet.

57. The method of claim 52, wherein the droplet is a sample droplet having a target antigen as a sample therein.